Various fats and fatty oils comprise triglycerides of fatty acids. Many fatty acids, particularly polyunsaturated fatty acids, are difficult to synthesize and can only be obtained by extraction from natural fats or fatty oils in which they naturally occur. Many of these unsaturated fatty acids are known to have therapeutic potential. Others are thought to have undesirable effects. Furthermore, the fatty acids having therapeutic properties must be in a particular cis-trans isomeric form. Processes for obtaining such unsaturated fatty acids in pure form and high quantity, without cis-trans conversion, have been long sought.
Two particular polyunsaturated fatty acids which have been shown to have therapeutic efficacy, and which are difficult to obtain in pure form and high quantities, are (all-Z)5,8,11,14,17-eicosapentaenoic acid, hereinafter referred to as EPA, and (all-Z)-4,7,10,13,16,19-docosahexaenoic acid, hereinafter referred to as DHA. Both EPA and DHA are known to be precursors in the biosynthesis of prostaglandin PGE.sub.3.
British Pat. Nos. 1,604,554 and 2,033,745 disclose thrombo-embolic conditions. British patent publication No. 2,148,713, to the present inventor, also discloses the use of EPA and/or DHA, in combination with other specific fatty acids, for reduction of serum cholesterol and triglyceride levels.
It has heretofore been very difficult to obtain pure EPA and DHA since the main source of these fatty acids is the fats and oils of marine animals, such as the mackerel, sardine or cod, and vegetable oils, such as linseed oil, either as itself or in the form of a derivative, such as a triglyceride. Unfortunately, other fatty acids are always present in larger amounts. Since EPA and DHA are known to have medical effectiveness, highly pure EPA and DHA are required in large amounts in order to conduct clinical studies.
Previous methods for extraction of EPA, DHA and other useful polyunsaturated fatty acids from their triglycerides, have not been satisfactory for the production of highly pure fatty acids. The term "purity" is used here to mean not only in the sense of being separated from all other fatty acids of different chain lengths and different number and placement of unsaturations, but also purity of the particular cis-trans structure. Prior art methods not only did not yield sufficient purity, but in many cases also required such extreme physical and chemical conditions as to cause some degree of degradation of the fatty acids, formation of peroxides, and/or conversion of at least some of the cis- bonds to the trans- form. Furthermore, many prior art processes use materials which are not on the Generally Recognized As Safe (GRAS) list of the U.S. Food and Drug Administration. In order for the final product to be used in foods and drugs it is important that there be no non-GRAS substances in the final product.
One prior art method of purifying EPA is disclosed in U.S. Pat. No. 4,377,526. In this patent, a mixture of fatty acids containing EPA is treated with urea in order to remove saturated fatty acids and fatty acids of lower unsaturation. The resultant solution is then subjected to fractional distillation in order to obtain higher yields of EPA. The fractional distillation, however, requires a temperature of at least 180.degree. C. over a period of at least 40 minutes. The best purity which can be obtained by this method set forth in any of the examples of this patent is 92.9%. Furthermore, it has been discovered that a substantial amount of the EPA produced by this method, in some cases as high as 20%, has some degree of cis-trans conversion. Any amount of the trans- form of EPA is strictly undesirable for food or pharmaceutical use.
Abu-Nasr, A. N. et al, J. Am. Oil Chemists Soc. 31, 41-45 (1954) discloses isolation of methyl eicosapentaenoate and ethyl docosahexaenoate starting with cod liver oil acids, using preliminary concentration by precipitation of the pure complexes followed by chromatographic separations. This technique does not give high enough purity and chromatographic separations require undesirably high amounts of solvent.
Teshima, S. et al, Bulletin of the Japanese Society of Scientific Fisheries, 44 (8) 927 (1978) describe a method for isolation of EPA and DHA from squid liver oil by saponifying with ethanolic potassium hydroxide, extracting the fatty acids with ether and methylating. The crude fatty acid methyl ester is purified by column chromatography on Silica Gel 60 and then the EPA is separated from the DHA by column chromatography on a mixture of silver nitrate and silica gel. The problem with this technique is tha there are often traces of silver left in the final product which is extremely undesirable in a food or pharmaceutical for human consumption. Furthermore, very high amounts of solvent are necessary in order to conduct the column chromatography. Other disclosures of the use of column chromatography to separate and purify, to some extent, EPA are described in Japanese Kokai No. 56-115736 and Russian 973,128.
Another prior art method of obtaining high purity EPA is disclosed in British patent publication No. 2,148,713. This publication describes a process in which the double bonds of the unsaturated fatty acids, in a mixture of fatty acids, are iodinated, followed by saponification of the iodinated oil, extraction of the fatty acids from the saponification mixture, methylation of the iodinated fatty acids, separation of the fatty acids by column chromatography, and then deiodination of the desired fractions. This process permits excellent resolution of the fatty acids upon eventual column chromatography, and protects the fatty acids from oxidation during processing. When used to separate EPA from a natural source of EPA, such as cod liver oil, a yield of over 90% and a purity of 96-100% may be obtained. It has been found, however, that a substantial amount of cis-trans conversion occurs in the course of this process, so that the product obtained of a purity of 96-100% is not pure all cis- EPA. Furthermore, iodine is not on the list of GRAS materials.
Various techniques of separation have been used with respect to fatty acids in general. Among these known techniques are separation by means of urea complexes and separation by means of low temperature fractional crystallization.
The separation technique utilizing urea involves the formation of crystalline inclusion compounds, also called adducts or complexes, between urea and various straight chain organic compounds. Inclusion compounds are combinations of two or more compounds, one of which is contained within the crystalline framework of the other. The components of an inclusion compound are each capable of separate existence, and they have no obvious way of uniting chemically. They are held together by secondary valence forces and by hydrogen bonds. Inclusion compounds differ from the conventional hydrogen-bonded systems, however, because the size and shape of the "host" and "guest" molecules are critically important in the former but may play little or no part in the latter.
It is known that the more saturated a long chain fatty acid, the more readily is a urea complex formed. Thus, saturated and most mono-unsatuated compounds may be separated from polyunsaturated compounds by treatment with urea. The techniques of separation by urea complexes are described in great detail in Swern, D. "Techniques of Separation: E. Urea Complexes" in Fatty Acids: Their Chemistry, Properties, Production and Uses, edited by Klare S. Markley, Part 3, pages 2309-2358, Interscience Publishers, New York, 1964, the entire contents of these pages being hereby incorporated by reference.
Low temperature fractional distillation has been used for separation of fatty acids and monoesters, and also for the separation of glycerides of natural fats and other lipid substances. The technique involves dissolving the fatty acids in a solvent and then lowering the temperature in order to cause crystallization of the various fatty acids from the solvent. Often sub-zero (.degree.C.) temperatures are used. This technique has many limitations, however. It is difficult to obtain great degrees of purity when separating mixtures of many fatty acids, and there are problems of mutual solubility of various acids. The techniques and limitations of low temperature crystallization are described in detail in Markley, K. S., "Techniques of Separation: A. Distillation, Salt Solubility, Low-Temperature Crystallization" at pages 2081-2123 of the above cited Markley text, the entire contents of which are hereby incorporated herein by reference.
Privett, O. S. et al, J.Am. Oil Chemist. Soc., 36, 443-449 (1959), describe a technique involving a combination of low temperature crystallization and urea complexes. In the analysis of pork liver lipids, low temperature fractionation was first used to obtain two fractions and the filtrate was subjected to fractionation from methanol via the urea inclusion compounds. Each fraction and the filtrate was esterified and distilled, and the various distillates subjected to analysis. Swern, D. et al, J. Am. Oil Chemist. Soc., 29, 614-615 (1952) disclose precipitating urea complexes from olive oil to remove saturated and mono-unsaturated compounds and then subjecting the acids or esters isolated from the urea complexes to low temperature crystallization and to fractional dittillation in order to produce oleic acid at 97-99% purity. Swern, D. et al, J.Am. Oil Chemist. Soc., 29, 431-434 (1952) and U.S. Pat. No. 2,838,480 isolate oleic acid from tallow, grease or red oil in 80-95% purity by first separating saturated acids by crystallization from 90% methanol at 0.degree. C., followed by addition of urea to the filrate to precipitate the adduct of oleic acid at room temperature.
None of the prior art techniques, however, suggests a method for separating EPA and DHA from marine animal oil in substantially 100% purity, without cis-trans conversion, using only materials generally recognized as safe and capable of being used in an industrial process. Methods for separating other specific polyunsaturated fatty acids from natural sources in very high purity are also long-sought in the prior art.